Two cGI PDE subfamilies (cGIP1 and cGIP2), products of distinct but related genes, have recently been cloned in our laboratory from rat (R) adipose tissue and human (H) cardiac cDNA libraries. Deduced sequences of the conserved domains of RcGIP1 and HcGIP2 are very similar to each other, except within a sequence of 44 amino acids not found in other PDE gene families. Although this cGI PDE insertion distinguishes cGI PDE conserved domains from those of other PDE families, it may also be a distinguishing characteristic of cGI PDE subfamilies. We have made a series of deletion mutants of RcGIP1 and HcGIP2 in order to define the catalytic domain boundaries and to determine the importance of the cGI PDE insertion for cGI PDE activity and inhibition by cAMP, a selective Type III cGI PDE inhibitor (cilostamide) and rolipram (a selective Type IV cAMP-specific PDE inhibitor). The active enzymes are relatively sensitive to inhibition by cilostamide and insensitive to rolipram. The differences in the 44 amino acid insertion in the conserved domain are not apparently reflected in different inhibitor sensitivities of RcGIP1 and HcGIP2 PDEs. These and other results indicate that the catalytic domains of cGI PDEs include the conserved region plus additional N- and C-terminal amino acids.